Simultaneous irrigation and negative pressure wound therapy enhances wound healing and reduces wound bioburden in a porcine model Kathryn Davis, PhD; Jessica Bills, BS; Jenny Barker, PhD; Paul Kim, DPM; Lawrence Lavery, DPM, MPH Department of Plastic Surgery, University of Texas Southwestern Medical Center, Dallas, Texas
Reprint requests: Dr. K. Davis, Department of Plastic Surgery, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, F4.310A, Dallas, TX 75390-8560, USA. Tel: +1 214 648 9159; Fax: +1 214 648 2550; Email: [email protected] Manuscript received: May 5, 2013 Accepted in final form: July 16, 2013 DOI:10.1111/wrr.12104
ABSTRACT Infected foot wounds are one of the most common reasons for hospitalization and amputation among persons with diabetes. The objective of the study was to investigate a new wound therapy system that employs negative pressure wound therapy (NPWT) with simultaneous irrigation therapy. For this study, we used a porcine model with full-thickness excisional wounds, inoculated with Pseudomonas aeruginosa. Wounds were treated for 21 days of therapy with either NPWT, NPWT with simultaneous irrigation therapy using normal saline or polyhexanide biguanide (PHMB) at low or high flow rates, or control. Data show that NPWT with either irrigation condition improved wound healing rates over control-treated wounds, yet did not differ from NPWT alone. NPWT improved bioburden over control-treated wounds. NPWT with simultaneous irrigation further reduced bioburden over control and NPWT-treated wounds; however, flow rate did not affect these outcomes. Together, these data show that NPWT with simultaneous irrigation therapy with either normal saline or PHMB has a positive effect on bioburden in a porcine model, which may translate clinically to improved wound healing outcomes.
Negative pressure wound therapy (NPWT) has shown remarkable effects on wound healing in chronic wounds.1,2 Despite the fact that NPWT is widely embraced in clinical practice as the most commonly used advanced wound therapeutic device, the mechanism underlying its efficacy is still under investigation. It has been proposed that NPWT promotes drainage of excessive fluid and debris and induces mechanical forces on the wound edges that promote healing.3–5 In addition, other studies have suggested that NPWT decreases bacterial colony formation,5,6 decreases edema,6–8 increases immune response,6–9 and decreases permeability of vessels.6–10 Further, it is thought that NPWT promotes healing by secondary intention through increased angiogenesis and blood flow to the wound margins.6,11,12 Infusion chemotherapy as an adjunct to NPWT is a relatively new concept and is not yet widely embraced in clinical practice. There are no preclinical animal studies and few clinical studies that use infusion chemotherapy.13–16 Preliminary clinical evidence suggests that there is a synergistic effect when the wound is treated with an infusion during NPWT.13,14,17,18 There are a variety of agents and dosing parameters that have been used in combination with NPWT infusion. However, there are no studies that compare the efficacy of different irrigation solutions or evaluate NPWT with and without irrigation. The study presented here is a 2 × 2 study that investigates the effects of irrigation therapy with two different solutions at two different flow rates on wound healing in the context of NPWT. In addition to wound healing efficacy, the extent of bacterial infiltration in wounds (“bioburden”) was assessed over 21 days of treatment. Wound Rep Reg (2013) 21 869–875 © 2013 by the Wound Healing Society
MATERIALS AND METHODS Animals and surgical procedures
A porcine model was used for this study. Female pigs (6) with a weight between 90 and 120 lbs were purchased from Change of Pace (Aubrey, TX). Animals were kept in singly housed temperature-controlled environment at 61–81°F. During a minimum of 5 days acclimation and experimental procedures, pigs were fed standard porcine chow ad libitum (Harlan, Houston, TX). Animals were acclimated to the articulated arm that supported the therapeutic device for 7 days prior to surgery to ensure minimal distress. All animals were fasted 12 hours prior to surgery. Animals were anesthetized using (3 mg/kg) Telazol (Ft. Dodge Veterinaria, Ft. Dodge, IA) and xylazine (10 mg/kg) by intramuscular injection and were maintained on constant flow of an isoflurane/oxygen (1.5–3%) mixture via facemask prior to intubation. A presurgical injection of atropine (0.04 mg/kg) was also administered intramuscularly. Intravenous access was established for hydration with Lactated Ringer’s solution. A water blanket and Bair Hugger (Arizant Healthcare, Eden Prairie, NJ) were used to maintain body temperature. The dorsum of the animal, between the scapula and posterior iliac crests, was cleaned, shaved, and treated with Veet (Reckitt Benckiser, Parsippany, NJ) for hair removal. The animal was disinfected with chlorhexadine and 70% alcohol three times, and then draped with sterile surgical drapes. Aseptic technique was used to place 6 2.5 cm diameter full thickness wounds to the dorso-lateral surface of the animal. Care of all animals and procedures were approved by the UT Southwestern Medical Center Institutional Animal Care and Use Committee. 869
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Table 1. Experimental therapy conditions Therapy Control NPWT Sal Lo Sal Hi PHMB Lo PHMB Hi
NPWT No −125 mm −125 mm −125 mm −125 mm −125 mm
Hg Hg Hg Hg Hg
Infusion soln
Flow rate
N /A N/A Normal saline Normal saline PHMB (0.1%) PHMB (0.1%)
N/A N/A ∼15 cc/hour ∼40 cc/hour ∼15 cc/hour ∼40 cc/hour
Hi, high; Lo, low; NPWT, negative pressure wound therapy; PHMB, polyhexanide biguanide; Sal, saline; soln, solution.
Bacterial inoculation
After wounding, each surgical site was inoculated with approximately 500 colony-forming units (CFUs) of Pseudomonas aeruginosa (American Type Culture Collection 6538), suspended in normal saline. The wounds were packed with saline-moistened gauze and covered with Tegaderm (3M, St. Paul, MN). Animals were allowed to recover from anesthesia and then placed in their cages. Inoculated wounds were allowed to incubate for 3 days prior to the initiation of therapy. Therapy application and administration
Three days after inoculation, dressings were removed, wounds were assessed (see below), and dressings for each therapeutic cohort were applied. Six (6) therapeutic groups were investigated in this study, each represented once per pig (Table 1). The wound position for each therapeutic group was randomized on each pig to prevent possible positional differences in wound healing that may have otherwise confounded the results. NPWT was administered using the Quantum™ (Innovative Therapies, Inc., Pompano Beach, FL) at-125 mm Hg of pressure. All wounds were dressed with polyurethane foam (Black Foam, Innovative Therapies, Inc.) using the mushroom technique. Skin was prepped with Allkare wipes (Convatec, Inc., Skillman, NJ) and Stomahesive paste (Convatec, Inc). Tegaderm layers were placed over the skin around the wound to prevent maceration. A polyurathane foam plug was cut to fill the wound volume. A larger block of foam was placed over the plug and Tegaderm was applied. For the NPWT condition, the Quantum connection was applied. For all irrigation conditions, both the Quantum and SpeedConnect Tubing (Innovative Therapies, Inc, Pompano Beach, FL) were applied. Infusion systems, with either normal saline or polyhexanide biguanide (PHMB) (Prontosan [0.01%] (B-Braun, Bethlehem, PA), were set to continuously infuse either 15 cc/hour (low flow) or 40 cc/hour (high flow) through the wound. Actual infusion volumes were attained every 12 hours (determined by subtracting the remaining volume from the initial fill volume) and were calculated for the entirety of the experiment. Therapies were kept the same for each wound for the duration of the study. Freedom of motion therapy arm
This study utilizes an articulated freedom-of-motion therapy arm that attaches to the side of the animal cage, has movement 870
in all three dimensions, and allows the therapy mechanism to swivel with the pig. The animal connects to the arm using a pin-locking mechanism with the d-ring on the dorsal aspect of a standard dog harness, which allows for quick release in the event that the animal needs to be unattached. The freedomof-motion arm is a novel method to study wound healing in a porcine model for extended periods of time under conditions of less physiologic stress to the animal. This contrasts previous studies that have investigated wound healing in restrained animals, which results in increased physiologic stress and subsequently, shorter study times. The freedom-of-motion therapy delivery system allows accurate and reliable delivery of wound healing therapeutics to a large animal without undue stress or intrusion. Dressing change, wound assessment, and sacrifice
Dressings were changed at days 4, 8, 11, 14, and 18 after therapy initiation. All animals were fasted for 12 hours prior to dressing change. Animals were anesthetized using Telazol (3 mg/kg) and xylazine (10 mg/kg) by intramuscular injection and maintained on a constant flow of isoflurane/oxygen via facemask. The skin surrounding the wound sites was cleaned with saline-soaked gauze and treated with Veet for hair removal. Wounds were assessed for inflammation in or around the wound bed, excessive discharge, even granulation tissue formation, induration, skin maceration, and general appearance (color, bleeding before or during debridement). After assessment, wounds were lightly debrided and dressings replaced. Photos were taken of each wound at each dressing change. Photos were scaled and analyzed by NIH ImageJ Bethesda, MD to obtain wound area. Wound area was calculated as percent change from surgical incision area. Animals were anesthetized with an intramuscular injection of Telazol (6 mg/kg) and sacrificed with Euthasol (Verbac AH, Inc, Ft. Worth, TX) (120 mg/kg) injection intravenously. Analyses of wound bioburden
At the initiation of therapy and at sacrifice (day 21), wound dressings were removed and using a scalpel, scrapings of each wound bed were taken and snap-frozen on dry ice. Samples were processed by Pathogenius, Inc. (Lubbock, TX) and analyzed for levels of Pseudomonas aeruginosa. The realtime PCR assay for all the panel organisms utilizes the LightCycler 480. Real-time polymerase chain reaction (PCR) detects the different bacterial and fungal DNA strains present in the specimen and the concentration of those organisms can be precisely calculated from the amplification results. The PCR exploits the 5′ nuclease activity of DNA polymerase to cleave a TaqMan probe during PCR extension. The TaqMan probe contains a reporter dye at the 5′ end of the probe and a quencher dye at the 3′ end of the probe. During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. The quantitation is based off a standard curve for each analyte and the cycle threshold or where the fluorescence detected crosses over the baseline. The copy per sample is then calculated to a standard amount per mg based on weight and extraction efficiency for each individual analyte. Wound Rep Reg (2013) 21 869–875 © 2013 by the Wound Healing Society
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Statistics
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A
The data are presented as mean ± SEM. After confirming normal distribution of data, comparisons between conditions were made by the unpaired two-tailed Student’s t-test; repeated-measures analysis of variance were used to compare changes over time between conditions. A p-value less than 0.05 is considered to be statistically significant. Significance to control is denoted by a (*) and significance to NPWT is denoted by a (#).
RESULTS Wound healing with irrigation therapy using normal saline or PHMB is equivalent to NPWT
Every 12 hours, irrigation volumes were monitored to assure equivalent treatment of wounds in each condition. Average daily irrigation volumes were calculated and showed equivalences between the irrigation solutions tested for low and high flow rate (Figure 1A). Additionally, the hourly administration volume was also noted above each bar. Wounds were assessed at 0, 4, 8, 11, 14, and 21 days after therapy initiation. Wound appearance was normal for all therapies tested (representative images shown in Figure 1B). There were no major instances of wound induration or inflammation of the wound margins for those wounds receiving NPWT or the irrigation therapy conditions. Additionally, no differences in skin maceration or granulation tissue integrity were observed in these groups. The control-treated conditions did show greater occurrence of uneven granulation tissue deposition and irregular wound margins. Wound bleeding did not differ between any of the conditions. Wound area was calculated for all time points. The controltreated wounds had delayed healing compared with both NPWT and irrigation conditions (Figure 2A). However, the efficacy of wound healing did not differ between NPWT and the irrigation conditions. At day 21, NPWT and all irrigation conditions had wound areas that were found to be 50% smaller than control wound areas (Figure 2B). However, the healing induced by high or low flow irrigation using saline or PHMB was equivalent to that of NPWT. At therapy day 4, wound depth and volume were reduced with NPWT relative to control treated wounds (Figure 3A and B). Irrigation therapy with normal saline or PHMB also improved wound depth and volume over control-treated wounds. However, neither irrigation solution or flow rate showed improvement over NPWT alone. By therapy day 7, almost all wounds in the experiment had filled (34 out of 36). Together, these data show that NPWT and NPWT with irrigation therapy accelerate wound healing over control conditions. However, the effects of NPWT and NPWT with irrigation on wound healing are equivalent. Infusion therapy with normal saline or PHMB results in a significant reduction in P. aeruginosa bioburden
Wounds were inoculated with P. aeruginosa for 3 days prior to therapy initiation. Levels of P. aeruginosa were measured at day 0 and day 21 after therapy initiation from samples obtained during wound debridement (Figure 4). With Control treatment, P. aeruginosa bioburden increased approximately Wound Rep Reg (2013) 21 869–875 © 2013 by the Wound Healing Society
B
Figure 1. Wound therapy conditions and wound outcomes. A. Average daily instillation volumes for all conditions with notations of hourly volumes administered. B. Representative day 21 wound images. Hi, high; Lo, low; NPWT, negative pressure wound therapy; PHMB, polyhexanide biguanide; Sal, saline; soln, solution.
5 × 107 (CFU) over the 21-day time course. This proliferation was substantially reduced with application of NPWT or NPWT with irrigation. Irrigation therapy with high flow saline or high and low flow PHMB resulted in reduced bioburden over NPWT alone. The reduction in P. aeruginosa with low flow saline was not statistically different than with NPWT (p = 0.0687). These data suggest that the addition of irrigation therapy to NPWT reduces wound bioburden over NPWT alone.
DISCUSSION This study was designed to test the effectiveness of irrigation therapy coupled with continuous NPWT for wound healing 871
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A
contrasts the intermittent therapeutic strategy employed by other NPWT irrigation devices. The 2 × 2 study design (two different solutions and two different flow rates) allowed us to determine that irrigation therapy improves wound healing over control-treated wounds, regardless of irrigation solution or flow rate. However, irrigation therapy does not improve wound healing over NPWT alone, suggesting that the direct effects of irrigation therapy on wound healing are minimal in an acute wound model and that NPWT is adequate for accelerated wound closure in this animal model. Bacterial load or “bioburden” contributes to wound chronicity. However, our ability to accurately evaluate the extent of bacterial infiltration in the clinical setting is poor. It has been suggested that wounds with >105 organisms per gram of tissue are associated with delayed wound healing in chronic
B
A
Figure 2. Wound area is improved with NPWT or NPWT with instillation therapy. A. Average wound area for each condition over time. B. Day 21 wound area for each condition. Data are presented as % of Day 0 measurement. Significance to control is denoted by a (*). NPWT, negative pressure wound therapy. Hi, high; Lo, low; PHMB, polyhexanide biguanide; Sal, saline; soln, solution.
and bioburden reduction. To date, there are no published, controlled, preclinical studies that evaluate irrigation therapy and its effectiveness. To perform these experiments, a novel therapy arm was developed to facilitate long-term, constant therapy in a porcine model without restrictive, physiologic stress to the animal. The data presented here indicate that wound-healing efficacy is equivalent between NPWT and NPWT with irrigation therapy. Both groups were found to be superior to controltreated wounds. There were no adverse events noted with any of the irrigation therapy conditions tested. The cohorts with increased rate of flow did not experience skin maceration or problems with granulation tissue formation. The ease of dressing application between NPWT and NPWT with irrigation therapy was equivalent and the integrity of the dressings between dressing changes was maintained. The irrigation therapy tested in this study was continuously applied, which 872
B
Figure 3. Wound depth and volume are improved with NPWT or NPWT with instillation therapy. A. Wound depth at posttherapy day 4. B. Wound volume at posttherapy day 4. Data are presented as % of Day 0 measurement. Significance to control is denoted by a (*). NPWT, negative pressure wound therapy. Hi, high; Lo, low; PHMB, polyhexanide biguanide; Sal, saline; soln, solution. Wound Rep Reg (2013) 21 869–875 © 2013 by the Wound Healing Society
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Figure 4. NPWT and NPWT with instillation therapy improve Pseudomonas aeruginosa bioburden. Quantitative cultures using qPCR were performed from wound scrapings. Data are presented as % of Day 0 measurement. Significance to control is denoted by a (*) and significance to NPWT is denoted by a (#). NPWT, negative pressure wound therapy. Hi, high; Lo, low; PHMB, polyhexanide biguanide; Sal, saline; soln, solution.
wounds.19,20 In a study by Xu et al., the rate of healing had a strong inverse relationship with CFUs. For every log order of CFUs there was a 44% delay in wound healing.21 This suggests that therapies aimed at reducing the burden of bacterial infiltration in wounds may facilitate healing. NPWT is used extensively to treat infected wounds. Experimental studies in pigs have shown a reduction in CFUs per gram from 108 to 106 with NPWT22 as well as a reduction in biofilm formation.22 Similarly, our data show that treatment with NPWT reduces pseudomonas colonization and enhances wound healing compared with control treatment. It is possible that enhanced healing was in part due to reduced bacterial load; however, the further reduction in pseudomonas levels seen with irrigation therapy did not correlate with greater reduction in wound area. Therefore, it is possible that reduction in wound healing is independent of the reduction in pseudomonas colonization. Alternatively, three hypotheses may explain why this model underestimates the effect of bacterial infiltration on wound healing. First, patients with chronic wounds, particularly those with diabetes, are more sensitive to infection than the well population. This is due to impaired vascular access and glucose-mediated inhibitory effects on neutrophils that are normally required to combat infection.23–27 Conversely, pigs are highly resistant to infection. Therefore, it is possible that the levels of pseudomonas used in our model were not proportionally high enough to recreate the inhibition of wound healing that may occur in the clinical setting due to bacterial infiltration. Another hypothesis is that NPWT alone reduces bioburden below a threshold that delays wound healing in pigs and that the further reduction of pseudomonas induced by irrigation therapy has little effect on wound healing in pigs. Lastly, while helpful and informative, the wounds studied in these experiments are acute wounds in healthy young animals whose biology differs significantly from complex chronic wounds in humans with Wound Rep Reg (2013) 21 869–875 © 2013 by the Wound Healing Society
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multiple comorbidities. Thus, the data presented here may understate the potential effects that could be observed with chronic wounds in compromised patients. In fact, animal data seem to sharply contrast clinical studies in high-risk, compromised patients. For instance in a randomized clinical study of diabetic foot wounds, Armstrong and Lavery reported that recurrent clinical infections were more common in patients that received NPWT compared with “standard wound therapy” (NPWT 16.9% vs. 9.4%); however, the difference was not statistically significant. Despite more clinical infections in this study, a significantly higher proportion of wounds in the NPWT group healed within 16 weeks (56% vs. 39%, p = 0.04).28 There are many irrigation products designed to decrease bacteria in wounds13,14,16 and several of these have been used as irrigation solution in retrospective NPWT studies. The choice of chemotherapeutic agents requires a trade-off between the ability to kill bacteria and the risk of damaging the wound bed and impeding the healing process. PHMB seems to be a very attractive choice to combine with NPWT because it has strong antibacterial properties with little cell toxicity. In fact, clinical studies have shown that it enhances wound healing.29 PHMB is a strong base and interacts with acidic phospholipids in the cell membrane, leading to increased permeability and cell death.30 PHMB has a broad antimicrobial spectrum, including Grampositive and Gram-negative bacteria as well as biofilmforming organisms.31 A greater than 5 log 10 reduction after 5 minutes of application is achieved with 0.02% PHMB against Staphylococcus aureus, Escherichia coli, Enterococcus faecium, P. aeruginosa, and Candida albicans.32 PHMB is classified as “practically nontoxic,” based on animal studies of skin and eye irritation.33 Kramer et al.29 compared PHMB and octenidine and Ringer’s lactate solution in superficial 20 mm diameter wounds in pigs. There were no differences in histology or tissue compatibility in the treatment groups. There was more wound contracture and faster wound healing in PHMD patients compared with the other treatments. SchmitNeuerburg conducted a double-blinded randomized clinical trial in contaminated wounds and compared 0.2% PHMB (n = 45) and Ringer’s lactate solution (n = 35). The PHMB group had better wound healing and faster reduction of Grampositive infections.34 Valenzuela and Perucho35 evaluated PHMB in a randomized controlled trial in chronic wounds (n = 142) comparing “standard of care” with 0.1% PHMB gel. Patients in the PHMB groups demonstrated reversal of positive cultures (p = 0.004), decreased wound area (p = 0.013), and increased granulation tissue (p = 0.001) compared with standard of care treatments. In the study presented here, there was a trend for PHMB treatment to improve bioburden over normal saline therapy; however, this difference was not statistically different. It is unknown if the difference in these results and those published previously are due to treatment paradigm and delivery method or if the wound healing effect of PHMB that has been observed with topical treatment is lost next to the wound-healing effects of NPWT. To this end, it is possible that in a wound with more significant contamination, PHMB may present better outcomes than normal saline. However, additional studies would be needed to determine this. In this study, an improvement in bioburden was observed with both normal saline and PHMB infusion. This effect persisted with the use of either high or low flow rate, suggesting that small volumes of wound flushing are equally as efficacious at reducing wound bioburden. Irrigation therapy 873
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with normal saline at a low flow rate did not statistically differ over NPWT alone when assessing pseudomonas growth; however, this may have been due to the small sample size and the error associated with that sample set. Clinically, a lower flow rate would be preferred as it reduces the cost of supplies and the time spent emptying collection canisters. This study was designed to test the effectiveness of simultaneous irrigation therapy to heal wounds and reduce bioburden. We demonstrate that irrigation therapy reduces pseudomonas colonization when compared with NPWT and control treatment. However, there was no enhanced wound healing observed with irrigation therapy over NPWT alone. These improvements in bioburden were not accompanied by adverse effects on the wound margin or granulation tissue bed. Given the data presented here, irrigation therapy coupled with NPWT may be an ideal candidate for clinical use aimed at reducing the sequelae associated with the infection of chronic wounds.
ACKNOWLEDGMENTS We thank the members of our lab, Deborah Noble, BS, Imelda Delgado, BS, and Jiying Huang, BS, who contributed to the studies in this manuscript. We also thank Sandra Berriman, PhD for critical contributions to this study and manuscript. Source of Funding: This research was funded through an educational grant from Innovative Therapies, Inc. Disclosures and Conflicts of Interest: Dr. Lavery has research grants from KCI, Osirus, Health Point, Thermotek, Integra, Glasko Smith and Kline, Convatec, and Innovative Therapies Inc. He is on the Speaker’s Bureau for Shire, KCI, and Innovative Technologies Inc. He is a Consultant/ Advisor for Innovative Therapies Inc., Pam Labs. He has Stock ownership in Diabetica Solutions and Prizm Medical and holds patents with Diabetica Solutions. Dr. Davis has grants from Convatec and Innovative Therapies, Inc. REFERENCES 1. Argenta LC, Morykwas MJ, Marks MW, DeFranzo AJ, Molnar JA, David LR. Vacuum-assisted closure: state of clinic art. Plast Reconstr Surg 2006; 117 (7 Suppl.): 127S–42S. 2. Banwell P, Teot L. Topical negative pressure (TNP): the evolution of a novel wound therapy. J Tissue Viability 2006; 16: 16–24. 3. Argenta LC, Morykwas MJ. Vacuum-assisted closure: a new method for wound control and treatment: clinical experience. Ann Plast Surg 1997; 38: 563–76; discussion 77. 4. Morykwas MJ, Argenta LC, Shelton-Brown EI, McGuirt W. Vacuum-assisted closure: a new method for wound control and treatment: animal studies and basic foundation. Ann Plast Surg 1997; 38: 553–62. 5. Morykwas MJ, Simpson J, Punger K, Argenta A, Kremers L, Argenta J. Vacuum-assisted closure: state of basic research and physiologic foundation. Plast Reconstr Surg 2006; 117 (7 Suppl.): 121S–6S. 6. Banwell PE. Topical negative pressure therapy in wound care. J Wound Care 1999; 8: 79–84. 7. Gouttefangeas C, Eberle M, Ruck P, Stark M, Muller JE, Becker HD, et al. Functional T lymphocytes infiltrate implanted polyvinyl alcohol foams during surgical wound closure therapy. Clin Exp Immunol 2001; 124: 398–405.
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31. Rosin M, Welk A, Bernhardt O, Ruhnau M, Pitten FA, Kocher T et al. Effect of a polyhexamethylene biguanide mouthrinse on bacterial counts and plaque. J Clin Periodontol 2001; 28: 1121–6. 32. Muller G, Kramer A. Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity. J Antimicrob Chemother 2008; 61: 1281–7. doi: 10.1093/jac/dkn125. 33. Hubner NO, Kramer A. Review on the efficacy, safety and clinical applications of polihexanide, a modern wound antiseptic. Skin Pharmacol Physiol 2010; 23 (Suppl.): 17–27. 34. Schmit-Neuerburg KP, Bettag C, Schlickewei W, Fabry W, Hanke J, Renzing-Kohler K, et al. [Effectiveness of an improved antiseptic in treatment of contaminated soft tissue wounds]. Der Chirurg; Zeitschrift fur alle Gebiete der operativen Medizen. 2001; 72: 61–71. 35. Valenzuela AR, Perucho NS. [The effectiveness of a 0.1% polyhexanide gel]. Rev Enferm. 2008; 31: 7–12.
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Reprint requests: Dr. K. Davis, Department of Plastic Surgery, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, F4.310A, Dallas, TX 75390-8560, USA. Tel: +1 214 648 9159; Fax: +1 214 648 2550; Email: [email protected] Manuscript received: May 5, 2013 Accepted in final form: July 16, 2013 DOI:10.1111/wrr.12104
ABSTRACT Infected foot wounds are one of the most common reasons for hospitalization and amputation among persons with diabetes. The objective of the study was to investigate a new wound therapy system that employs negative pressure wound therapy (NPWT) with simultaneous irrigation therapy. For this study, we used a porcine model with full-thickness excisional wounds, inoculated with Pseudomonas aeruginosa. Wounds were treated for 21 days of therapy with either NPWT, NPWT with simultaneous irrigation therapy using normal saline or polyhexanide biguanide (PHMB) at low or high flow rates, or control. Data show that NPWT with either irrigation condition improved wound healing rates over control-treated wounds, yet did not differ from NPWT alone. NPWT improved bioburden over control-treated wounds. NPWT with simultaneous irrigation further reduced bioburden over control and NPWT-treated wounds; however, flow rate did not affect these outcomes. Together, these data show that NPWT with simultaneous irrigation therapy with either normal saline or PHMB has a positive effect on bioburden in a porcine model, which may translate clinically to improved wound healing outcomes.
Negative pressure wound therapy (NPWT) has shown remarkable effects on wound healing in chronic wounds.1,2 Despite the fact that NPWT is widely embraced in clinical practice as the most commonly used advanced wound therapeutic device, the mechanism underlying its efficacy is still under investigation. It has been proposed that NPWT promotes drainage of excessive fluid and debris and induces mechanical forces on the wound edges that promote healing.3–5 In addition, other studies have suggested that NPWT decreases bacterial colony formation,5,6 decreases edema,6–8 increases immune response,6–9 and decreases permeability of vessels.6–10 Further, it is thought that NPWT promotes healing by secondary intention through increased angiogenesis and blood flow to the wound margins.6,11,12 Infusion chemotherapy as an adjunct to NPWT is a relatively new concept and is not yet widely embraced in clinical practice. There are no preclinical animal studies and few clinical studies that use infusion chemotherapy.13–16 Preliminary clinical evidence suggests that there is a synergistic effect when the wound is treated with an infusion during NPWT.13,14,17,18 There are a variety of agents and dosing parameters that have been used in combination with NPWT infusion. However, there are no studies that compare the efficacy of different irrigation solutions or evaluate NPWT with and without irrigation. The study presented here is a 2 × 2 study that investigates the effects of irrigation therapy with two different solutions at two different flow rates on wound healing in the context of NPWT. In addition to wound healing efficacy, the extent of bacterial infiltration in wounds (“bioburden”) was assessed over 21 days of treatment. Wound Rep Reg (2013) 21 869–875 © 2013 by the Wound Healing Society
MATERIALS AND METHODS Animals and surgical procedures
A porcine model was used for this study. Female pigs (6) with a weight between 90 and 120 lbs were purchased from Change of Pace (Aubrey, TX). Animals were kept in singly housed temperature-controlled environment at 61–81°F. During a minimum of 5 days acclimation and experimental procedures, pigs were fed standard porcine chow ad libitum (Harlan, Houston, TX). Animals were acclimated to the articulated arm that supported the therapeutic device for 7 days prior to surgery to ensure minimal distress. All animals were fasted 12 hours prior to surgery. Animals were anesthetized using (3 mg/kg) Telazol (Ft. Dodge Veterinaria, Ft. Dodge, IA) and xylazine (10 mg/kg) by intramuscular injection and were maintained on constant flow of an isoflurane/oxygen (1.5–3%) mixture via facemask prior to intubation. A presurgical injection of atropine (0.04 mg/kg) was also administered intramuscularly. Intravenous access was established for hydration with Lactated Ringer’s solution. A water blanket and Bair Hugger (Arizant Healthcare, Eden Prairie, NJ) were used to maintain body temperature. The dorsum of the animal, between the scapula and posterior iliac crests, was cleaned, shaved, and treated with Veet (Reckitt Benckiser, Parsippany, NJ) for hair removal. The animal was disinfected with chlorhexadine and 70% alcohol three times, and then draped with sterile surgical drapes. Aseptic technique was used to place 6 2.5 cm diameter full thickness wounds to the dorso-lateral surface of the animal. Care of all animals and procedures were approved by the UT Southwestern Medical Center Institutional Animal Care and Use Committee. 869
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Table 1. Experimental therapy conditions Therapy Control NPWT Sal Lo Sal Hi PHMB Lo PHMB Hi
NPWT No −125 mm −125 mm −125 mm −125 mm −125 mm
Hg Hg Hg Hg Hg
Infusion soln
Flow rate
N /A N/A Normal saline Normal saline PHMB (0.1%) PHMB (0.1%)
N/A N/A ∼15 cc/hour ∼40 cc/hour ∼15 cc/hour ∼40 cc/hour
Hi, high; Lo, low; NPWT, negative pressure wound therapy; PHMB, polyhexanide biguanide; Sal, saline; soln, solution.
Bacterial inoculation
After wounding, each surgical site was inoculated with approximately 500 colony-forming units (CFUs) of Pseudomonas aeruginosa (American Type Culture Collection 6538), suspended in normal saline. The wounds were packed with saline-moistened gauze and covered with Tegaderm (3M, St. Paul, MN). Animals were allowed to recover from anesthesia and then placed in their cages. Inoculated wounds were allowed to incubate for 3 days prior to the initiation of therapy. Therapy application and administration
Three days after inoculation, dressings were removed, wounds were assessed (see below), and dressings for each therapeutic cohort were applied. Six (6) therapeutic groups were investigated in this study, each represented once per pig (Table 1). The wound position for each therapeutic group was randomized on each pig to prevent possible positional differences in wound healing that may have otherwise confounded the results. NPWT was administered using the Quantum™ (Innovative Therapies, Inc., Pompano Beach, FL) at-125 mm Hg of pressure. All wounds were dressed with polyurethane foam (Black Foam, Innovative Therapies, Inc.) using the mushroom technique. Skin was prepped with Allkare wipes (Convatec, Inc., Skillman, NJ) and Stomahesive paste (Convatec, Inc). Tegaderm layers were placed over the skin around the wound to prevent maceration. A polyurathane foam plug was cut to fill the wound volume. A larger block of foam was placed over the plug and Tegaderm was applied. For the NPWT condition, the Quantum connection was applied. For all irrigation conditions, both the Quantum and SpeedConnect Tubing (Innovative Therapies, Inc, Pompano Beach, FL) were applied. Infusion systems, with either normal saline or polyhexanide biguanide (PHMB) (Prontosan [0.01%] (B-Braun, Bethlehem, PA), were set to continuously infuse either 15 cc/hour (low flow) or 40 cc/hour (high flow) through the wound. Actual infusion volumes were attained every 12 hours (determined by subtracting the remaining volume from the initial fill volume) and were calculated for the entirety of the experiment. Therapies were kept the same for each wound for the duration of the study. Freedom of motion therapy arm
This study utilizes an articulated freedom-of-motion therapy arm that attaches to the side of the animal cage, has movement 870
in all three dimensions, and allows the therapy mechanism to swivel with the pig. The animal connects to the arm using a pin-locking mechanism with the d-ring on the dorsal aspect of a standard dog harness, which allows for quick release in the event that the animal needs to be unattached. The freedomof-motion arm is a novel method to study wound healing in a porcine model for extended periods of time under conditions of less physiologic stress to the animal. This contrasts previous studies that have investigated wound healing in restrained animals, which results in increased physiologic stress and subsequently, shorter study times. The freedom-of-motion therapy delivery system allows accurate and reliable delivery of wound healing therapeutics to a large animal without undue stress or intrusion. Dressing change, wound assessment, and sacrifice
Dressings were changed at days 4, 8, 11, 14, and 18 after therapy initiation. All animals were fasted for 12 hours prior to dressing change. Animals were anesthetized using Telazol (3 mg/kg) and xylazine (10 mg/kg) by intramuscular injection and maintained on a constant flow of isoflurane/oxygen via facemask. The skin surrounding the wound sites was cleaned with saline-soaked gauze and treated with Veet for hair removal. Wounds were assessed for inflammation in or around the wound bed, excessive discharge, even granulation tissue formation, induration, skin maceration, and general appearance (color, bleeding before or during debridement). After assessment, wounds were lightly debrided and dressings replaced. Photos were taken of each wound at each dressing change. Photos were scaled and analyzed by NIH ImageJ Bethesda, MD to obtain wound area. Wound area was calculated as percent change from surgical incision area. Animals were anesthetized with an intramuscular injection of Telazol (6 mg/kg) and sacrificed with Euthasol (Verbac AH, Inc, Ft. Worth, TX) (120 mg/kg) injection intravenously. Analyses of wound bioburden
At the initiation of therapy and at sacrifice (day 21), wound dressings were removed and using a scalpel, scrapings of each wound bed were taken and snap-frozen on dry ice. Samples were processed by Pathogenius, Inc. (Lubbock, TX) and analyzed for levels of Pseudomonas aeruginosa. The realtime PCR assay for all the panel organisms utilizes the LightCycler 480. Real-time polymerase chain reaction (PCR) detects the different bacterial and fungal DNA strains present in the specimen and the concentration of those organisms can be precisely calculated from the amplification results. The PCR exploits the 5′ nuclease activity of DNA polymerase to cleave a TaqMan probe during PCR extension. The TaqMan probe contains a reporter dye at the 5′ end of the probe and a quencher dye at the 3′ end of the probe. During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. The quantitation is based off a standard curve for each analyte and the cycle threshold or where the fluorescence detected crosses over the baseline. The copy per sample is then calculated to a standard amount per mg based on weight and extraction efficiency for each individual analyte. Wound Rep Reg (2013) 21 869–875 © 2013 by the Wound Healing Society
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Statistics
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A
The data are presented as mean ± SEM. After confirming normal distribution of data, comparisons between conditions were made by the unpaired two-tailed Student’s t-test; repeated-measures analysis of variance were used to compare changes over time between conditions. A p-value less than 0.05 is considered to be statistically significant. Significance to control is denoted by a (*) and significance to NPWT is denoted by a (#).
RESULTS Wound healing with irrigation therapy using normal saline or PHMB is equivalent to NPWT
Every 12 hours, irrigation volumes were monitored to assure equivalent treatment of wounds in each condition. Average daily irrigation volumes were calculated and showed equivalences between the irrigation solutions tested for low and high flow rate (Figure 1A). Additionally, the hourly administration volume was also noted above each bar. Wounds were assessed at 0, 4, 8, 11, 14, and 21 days after therapy initiation. Wound appearance was normal for all therapies tested (representative images shown in Figure 1B). There were no major instances of wound induration or inflammation of the wound margins for those wounds receiving NPWT or the irrigation therapy conditions. Additionally, no differences in skin maceration or granulation tissue integrity were observed in these groups. The control-treated conditions did show greater occurrence of uneven granulation tissue deposition and irregular wound margins. Wound bleeding did not differ between any of the conditions. Wound area was calculated for all time points. The controltreated wounds had delayed healing compared with both NPWT and irrigation conditions (Figure 2A). However, the efficacy of wound healing did not differ between NPWT and the irrigation conditions. At day 21, NPWT and all irrigation conditions had wound areas that were found to be 50% smaller than control wound areas (Figure 2B). However, the healing induced by high or low flow irrigation using saline or PHMB was equivalent to that of NPWT. At therapy day 4, wound depth and volume were reduced with NPWT relative to control treated wounds (Figure 3A and B). Irrigation therapy with normal saline or PHMB also improved wound depth and volume over control-treated wounds. However, neither irrigation solution or flow rate showed improvement over NPWT alone. By therapy day 7, almost all wounds in the experiment had filled (34 out of 36). Together, these data show that NPWT and NPWT with irrigation therapy accelerate wound healing over control conditions. However, the effects of NPWT and NPWT with irrigation on wound healing are equivalent. Infusion therapy with normal saline or PHMB results in a significant reduction in P. aeruginosa bioburden
Wounds were inoculated with P. aeruginosa for 3 days prior to therapy initiation. Levels of P. aeruginosa were measured at day 0 and day 21 after therapy initiation from samples obtained during wound debridement (Figure 4). With Control treatment, P. aeruginosa bioburden increased approximately Wound Rep Reg (2013) 21 869–875 © 2013 by the Wound Healing Society
B
Figure 1. Wound therapy conditions and wound outcomes. A. Average daily instillation volumes for all conditions with notations of hourly volumes administered. B. Representative day 21 wound images. Hi, high; Lo, low; NPWT, negative pressure wound therapy; PHMB, polyhexanide biguanide; Sal, saline; soln, solution.
5 × 107 (CFU) over the 21-day time course. This proliferation was substantially reduced with application of NPWT or NPWT with irrigation. Irrigation therapy with high flow saline or high and low flow PHMB resulted in reduced bioburden over NPWT alone. The reduction in P. aeruginosa with low flow saline was not statistically different than with NPWT (p = 0.0687). These data suggest that the addition of irrigation therapy to NPWT reduces wound bioburden over NPWT alone.
DISCUSSION This study was designed to test the effectiveness of irrigation therapy coupled with continuous NPWT for wound healing 871
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A
contrasts the intermittent therapeutic strategy employed by other NPWT irrigation devices. The 2 × 2 study design (two different solutions and two different flow rates) allowed us to determine that irrigation therapy improves wound healing over control-treated wounds, regardless of irrigation solution or flow rate. However, irrigation therapy does not improve wound healing over NPWT alone, suggesting that the direct effects of irrigation therapy on wound healing are minimal in an acute wound model and that NPWT is adequate for accelerated wound closure in this animal model. Bacterial load or “bioburden” contributes to wound chronicity. However, our ability to accurately evaluate the extent of bacterial infiltration in the clinical setting is poor. It has been suggested that wounds with >105 organisms per gram of tissue are associated with delayed wound healing in chronic
B
A
Figure 2. Wound area is improved with NPWT or NPWT with instillation therapy. A. Average wound area for each condition over time. B. Day 21 wound area for each condition. Data are presented as % of Day 0 measurement. Significance to control is denoted by a (*). NPWT, negative pressure wound therapy. Hi, high; Lo, low; PHMB, polyhexanide biguanide; Sal, saline; soln, solution.
and bioburden reduction. To date, there are no published, controlled, preclinical studies that evaluate irrigation therapy and its effectiveness. To perform these experiments, a novel therapy arm was developed to facilitate long-term, constant therapy in a porcine model without restrictive, physiologic stress to the animal. The data presented here indicate that wound-healing efficacy is equivalent between NPWT and NPWT with irrigation therapy. Both groups were found to be superior to controltreated wounds. There were no adverse events noted with any of the irrigation therapy conditions tested. The cohorts with increased rate of flow did not experience skin maceration or problems with granulation tissue formation. The ease of dressing application between NPWT and NPWT with irrigation therapy was equivalent and the integrity of the dressings between dressing changes was maintained. The irrigation therapy tested in this study was continuously applied, which 872
B
Figure 3. Wound depth and volume are improved with NPWT or NPWT with instillation therapy. A. Wound depth at posttherapy day 4. B. Wound volume at posttherapy day 4. Data are presented as % of Day 0 measurement. Significance to control is denoted by a (*). NPWT, negative pressure wound therapy. Hi, high; Lo, low; PHMB, polyhexanide biguanide; Sal, saline; soln, solution. Wound Rep Reg (2013) 21 869–875 © 2013 by the Wound Healing Society
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Figure 4. NPWT and NPWT with instillation therapy improve Pseudomonas aeruginosa bioburden. Quantitative cultures using qPCR were performed from wound scrapings. Data are presented as % of Day 0 measurement. Significance to control is denoted by a (*) and significance to NPWT is denoted by a (#). NPWT, negative pressure wound therapy. Hi, high; Lo, low; PHMB, polyhexanide biguanide; Sal, saline; soln, solution.
wounds.19,20 In a study by Xu et al., the rate of healing had a strong inverse relationship with CFUs. For every log order of CFUs there was a 44% delay in wound healing.21 This suggests that therapies aimed at reducing the burden of bacterial infiltration in wounds may facilitate healing. NPWT is used extensively to treat infected wounds. Experimental studies in pigs have shown a reduction in CFUs per gram from 108 to 106 with NPWT22 as well as a reduction in biofilm formation.22 Similarly, our data show that treatment with NPWT reduces pseudomonas colonization and enhances wound healing compared with control treatment. It is possible that enhanced healing was in part due to reduced bacterial load; however, the further reduction in pseudomonas levels seen with irrigation therapy did not correlate with greater reduction in wound area. Therefore, it is possible that reduction in wound healing is independent of the reduction in pseudomonas colonization. Alternatively, three hypotheses may explain why this model underestimates the effect of bacterial infiltration on wound healing. First, patients with chronic wounds, particularly those with diabetes, are more sensitive to infection than the well population. This is due to impaired vascular access and glucose-mediated inhibitory effects on neutrophils that are normally required to combat infection.23–27 Conversely, pigs are highly resistant to infection. Therefore, it is possible that the levels of pseudomonas used in our model were not proportionally high enough to recreate the inhibition of wound healing that may occur in the clinical setting due to bacterial infiltration. Another hypothesis is that NPWT alone reduces bioburden below a threshold that delays wound healing in pigs and that the further reduction of pseudomonas induced by irrigation therapy has little effect on wound healing in pigs. Lastly, while helpful and informative, the wounds studied in these experiments are acute wounds in healthy young animals whose biology differs significantly from complex chronic wounds in humans with Wound Rep Reg (2013) 21 869–875 © 2013 by the Wound Healing Society
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multiple comorbidities. Thus, the data presented here may understate the potential effects that could be observed with chronic wounds in compromised patients. In fact, animal data seem to sharply contrast clinical studies in high-risk, compromised patients. For instance in a randomized clinical study of diabetic foot wounds, Armstrong and Lavery reported that recurrent clinical infections were more common in patients that received NPWT compared with “standard wound therapy” (NPWT 16.9% vs. 9.4%); however, the difference was not statistically significant. Despite more clinical infections in this study, a significantly higher proportion of wounds in the NPWT group healed within 16 weeks (56% vs. 39%, p = 0.04).28 There are many irrigation products designed to decrease bacteria in wounds13,14,16 and several of these have been used as irrigation solution in retrospective NPWT studies. The choice of chemotherapeutic agents requires a trade-off between the ability to kill bacteria and the risk of damaging the wound bed and impeding the healing process. PHMB seems to be a very attractive choice to combine with NPWT because it has strong antibacterial properties with little cell toxicity. In fact, clinical studies have shown that it enhances wound healing.29 PHMB is a strong base and interacts with acidic phospholipids in the cell membrane, leading to increased permeability and cell death.30 PHMB has a broad antimicrobial spectrum, including Grampositive and Gram-negative bacteria as well as biofilmforming organisms.31 A greater than 5 log 10 reduction after 5 minutes of application is achieved with 0.02% PHMB against Staphylococcus aureus, Escherichia coli, Enterococcus faecium, P. aeruginosa, and Candida albicans.32 PHMB is classified as “practically nontoxic,” based on animal studies of skin and eye irritation.33 Kramer et al.29 compared PHMB and octenidine and Ringer’s lactate solution in superficial 20 mm diameter wounds in pigs. There were no differences in histology or tissue compatibility in the treatment groups. There was more wound contracture and faster wound healing in PHMD patients compared with the other treatments. SchmitNeuerburg conducted a double-blinded randomized clinical trial in contaminated wounds and compared 0.2% PHMB (n = 45) and Ringer’s lactate solution (n = 35). The PHMB group had better wound healing and faster reduction of Grampositive infections.34 Valenzuela and Perucho35 evaluated PHMB in a randomized controlled trial in chronic wounds (n = 142) comparing “standard of care” with 0.1% PHMB gel. Patients in the PHMB groups demonstrated reversal of positive cultures (p = 0.004), decreased wound area (p = 0.013), and increased granulation tissue (p = 0.001) compared with standard of care treatments. In the study presented here, there was a trend for PHMB treatment to improve bioburden over normal saline therapy; however, this difference was not statistically different. It is unknown if the difference in these results and those published previously are due to treatment paradigm and delivery method or if the wound healing effect of PHMB that has been observed with topical treatment is lost next to the wound-healing effects of NPWT. To this end, it is possible that in a wound with more significant contamination, PHMB may present better outcomes than normal saline. However, additional studies would be needed to determine this. In this study, an improvement in bioburden was observed with both normal saline and PHMB infusion. This effect persisted with the use of either high or low flow rate, suggesting that small volumes of wound flushing are equally as efficacious at reducing wound bioburden. Irrigation therapy 873
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with normal saline at a low flow rate did not statistically differ over NPWT alone when assessing pseudomonas growth; however, this may have been due to the small sample size and the error associated with that sample set. Clinically, a lower flow rate would be preferred as it reduces the cost of supplies and the time spent emptying collection canisters. This study was designed to test the effectiveness of simultaneous irrigation therapy to heal wounds and reduce bioburden. We demonstrate that irrigation therapy reduces pseudomonas colonization when compared with NPWT and control treatment. However, there was no enhanced wound healing observed with irrigation therapy over NPWT alone. These improvements in bioburden were not accompanied by adverse effects on the wound margin or granulation tissue bed. Given the data presented here, irrigation therapy coupled with NPWT may be an ideal candidate for clinical use aimed at reducing the sequelae associated with the infection of chronic wounds.
ACKNOWLEDGMENTS We thank the members of our lab, Deborah Noble, BS, Imelda Delgado, BS, and Jiying Huang, BS, who contributed to the studies in this manuscript. We also thank Sandra Berriman, PhD for critical contributions to this study and manuscript. Source of Funding: This research was funded through an educational grant from Innovative Therapies, Inc. Disclosures and Conflicts of Interest: Dr. Lavery has research grants from KCI, Osirus, Health Point, Thermotek, Integra, Glasko Smith and Kline, Convatec, and Innovative Therapies Inc. He is on the Speaker’s Bureau for Shire, KCI, and Innovative Technologies Inc. He is a Consultant/ Advisor for Innovative Therapies Inc., Pam Labs. He has Stock ownership in Diabetica Solutions and Prizm Medical and holds patents with Diabetica Solutions. Dr. Davis has grants from Convatec and Innovative Therapies, Inc. REFERENCES 1. Argenta LC, Morykwas MJ, Marks MW, DeFranzo AJ, Molnar JA, David LR. Vacuum-assisted closure: state of clinic art. Plast Reconstr Surg 2006; 117 (7 Suppl.): 127S–42S. 2. Banwell P, Teot L. Topical negative pressure (TNP): the evolution of a novel wound therapy. J Tissue Viability 2006; 16: 16–24. 3. Argenta LC, Morykwas MJ. Vacuum-assisted closure: a new method for wound control and treatment: clinical experience. Ann Plast Surg 1997; 38: 563–76; discussion 77. 4. Morykwas MJ, Argenta LC, Shelton-Brown EI, McGuirt W. Vacuum-assisted closure: a new method for wound control and treatment: animal studies and basic foundation. Ann Plast Surg 1997; 38: 553–62. 5. Morykwas MJ, Simpson J, Punger K, Argenta A, Kremers L, Argenta J. Vacuum-assisted closure: state of basic research and physiologic foundation. Plast Reconstr Surg 2006; 117 (7 Suppl.): 121S–6S. 6. Banwell PE. Topical negative pressure therapy in wound care. J Wound Care 1999; 8: 79–84. 7. Gouttefangeas C, Eberle M, Ruck P, Stark M, Muller JE, Becker HD, et al. Functional T lymphocytes infiltrate implanted polyvinyl alcohol foams during surgical wound closure therapy. Clin Exp Immunol 2001; 124: 398–405.
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